Analysis of peripheral inflammatory T cell subsets and their effector function in patients with Birdshot Retinochoroiditis.

Birdshot Retinochoroiditis (BSRC) is a progressive non-infectious intraocular inflammation that affects choroid and retina. Inflammatory processes have adverse effects on vision by affecting photoreceptor-bearing cells that do not regenerate. This study aimed at characterizing inflammatory CD4+ and CD8+ T cell subsets in the peripheral blood of active and inactive BSRCs. Furthermore, we correlated phenotypical and functional immunological analyses with clinical data. We observed a slight increase of terminally differentiated effector memory CD8+ T cells expressing CD45RA (TEMRA) in blood of inactive, compared to active BSRCs. Moreover, we identified a trend for a decreased population of TH2 cells and increased TH1 frequencies in active BSRCs, a typical sign of ongoing autoimmune processes. Functional assays demonstrated severe and overall impairment of effector function of both, CD4+ and CD8+ inflammatory T cells, which might reflect T cell exhaustion. Although the eye is the main site of inflammation in BSRC, we observed altered T cell subset compositions in the peripheral blood, dependent on the disease status. Our results indicate that T cells may play a major role in BSRC pathology, although our cohort size is too limited for definitve conclusions. Future studies with larger BSRCs have to be performed.

Protocol for Efficient CRISPR/Cas9/AAV-Mediated Homologous Recombination in Mouse Hematopoietic Stem and Progenitor Cells.

Mutations that accumulate in self-renewing hematopoietic stem and progenitor cells (HSPCs) can cause severe blood disorders. To model such disorders in mice, we developed a CRISPR/Cas9/adeno-associated virus (AAV)-based system to knock in and repair genes by homologous recombination in mouse HSPCs. Here, we provide a step-by-step protocol to achieve high efficiency of gene knockin in mouse HSPCs, while maintaining engraftment capacity. This approach enables the functional study of hematopoietic disease mutations in vivo, without requiring germline mutagenesis. For complete details on the use and execution of this protocol, please refer to Tran et al. (2019).

Efficient CRISPR/Cas9-Mediated Gene Knockin in Mouse Hematopoietic Stem and Progenitor Cells.

Mutations accumulating in hematopoietic stem and progenitor cells (HSPCs) during development can cause severe hematological disorders. Modeling these mutations in mice is essential for understanding their functional consequences. Here, we describe an efficient CRISPR/Cas9-based system to knock in and repair genes in mouse HSPCs. CRISPR/Cas9 ribonucleoproteins, in combination with recombinant adeno-associated virus (rAAV)-DJ donor templates, led to gene knockin efficiencies of up to 30% in the Lmnb1 and Actb loci of mouse HSPCs in vitro. The targeted HSPCs engraft and reconstitute all immune cell lineages in the recipient mice. Using this approach, we corrected a neomycin-disrupted Rag2 gene. The Rag2-corrected HSPCs restore B and T cell development in vivo, confirming the functionality of the approach. Our method provides an efficient strategy to study gene function in the hematopoietic system and model hematological disorders in vivo, without the need for germline mutagenesis.